Accuracy and dynamic range of spatial image correlation and cross-correlation spectroscopy

¹ McGill University

^* To whom correspondence should be addressed. E-mail: paul.wiseman{at}mcgill.ca.

Submitted on December 3, 2004
Revised on January 31, 2005
Accepted on 11 April 2005

	Abstract

We present a comprehensive study of the accuracy and dynamic range of spatial image correlation spectroscopy (ICS) and image cross-correlation spectroscopy (ICCS). We use simulations to model laser scanning microscopy imaging of static sub-diffraction limit fluorescent proteins or protein clusters in a cell membrane. The simulation programs allow us to control the spatial imaging sampling variables, the particle population densities and interactions, and introduce and vary background and counting noise typical of what is encountered in digital optical microscopy. We systematically calculate how the accuracy of both image correlation methods depends on practical experimental collection parameters and characteristics of the sample. The results of this study provide a guide to appropriately plan spatial image correlation measurements on proteins in biological membranes in real cells. The data presented maps regimes where the spatial ICS and ICCS provide accurate results as well as clearly showing the conditions where they systematically deviate from acceptable accuracy. Finally, we compare the simulated data with standard confocal microscopy using live CHO cells expressing the epidermal growth factor receptor fused with green fluorescent protein (GFP/EGFR), in order to obtain typical values for the experimental variables that were investigated in our study. We used our simulation results to estimate a relative precision of 20% for the ICS measured receptor density of 64 µm^-2 within a 121x98 pixel sub-region of a single cell.

Key Words: accuracy, image correlation spectroscopy, image cross-correlation spectroscopy, precision, signal to noise

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